The interaction of neutrophils and mononuclear leukocytes with endothelium plays a pivotal role in leukocyte recruitment in acute and chronic inflammation, immune reactions, vascular injury and atherosclerosis. In the previous funding period, four major accomplishments occurred. (1) We identified a novel E-selectin dependent adhesion pathway in T cells subsets. (2) We determined that t-bet T cells have a defect in trafficking in vivo and in vitro related to impaired PSGL-1 function and failure to express CXCR4. (3) We developed a live cell fluorescence assay to resolve the temporal and spatial behavior of LFA-1 and ICAM-1 during leukocyte transmigration. (4) We developed an in vitro assay for non-junctional neutrophil transmigration. This proposal builds on this scientific progress and will consist of three interrelated specific aims that will focus on the role of endothelial cell ICAM-1 tail in leukocyte transmigration. Our working hypothesis is that during leukocyte adhesion, LFA-1 binds ICAM-1, triggering its association with the cytoskeleton, and this step functions in "outside in" signaling to promote ICAM-1 clustering around transmigrating leukocytes. Specific Aim 1 will elucidate the role of ICAM-1 cytoplasmic tail in leukocyte transmigration using biochemical and biophysical techniques as well as a newly developed live cell fluorescence imaging system and vascular endothelium that support both junctional and non-junctional transmigration. Specific Aim 2 will test the role of the ICAM-1 tail in chemoattractant driven leukocyte transmigration in an in vivo model of inflammation. Confocal intravital fluorescence imaging will visualize the sites of leukocyte transmigration (junctional or non-junctional route) using fluorescent markers of cell-cell junctions, and in parallel, determine whether the route of transmigration adversely effects vascular permeability. Specific Aim 3 will study the role of the ICAM-1 tail in an arterial endothelial cell model of inflammation in vitro. Endothelial monolayers of an arterial or venous phenotype will be created in vitro by applying a pulsatile shear stress in an arterial or venous waveform using a recently described cone-plate apparatus. The adhesive interactions and sites of transmigration of blood monocytes and memory T cells with the above conditioned endothelium will be examined. The information gained from these studies will help delineate the ICAM-1 dependent mechanisms underlying leukocyte transmigration and may thus define new targets for therapeutic intervention in immune and inflammatory diseases.